1. Field of the Invention
This invention relates to assay apparatus and procedures.
2. Introduction
In bead-based flow cytometry and similar assay procedures, the sample to be assayed is contacted with a multitude of particles. All the particles are coded for recognition purposes and contain analyte-interaction sites which interact selectively with one or more of the analytes in the sample. The particles and the sample are contacted for a time and under conditions such that the desired interaction takes place. The particles fall into different categories. In each category, all the particles                (a) have the same coding characteristic, and        (b) contain the same analyte-interaction sites.The coding characteristic and the analyte-interaction sites in each category are different from those in all the other categories.        
Before, during or after the contacting of the particles and the sample, the analytes and/or the sites which have interacted with the analytes are labeled with a signal label. The interaction between the analyte interaction sites can be of any kind, for example the formation of a covalent, coordinate or ionic bond, hybridisation of nucleotides, or enzymatic action. The term “analyte-bearing site” is used herein to denote any analyte interaction site which has interacted with an analyte, even if the analyte itself is not present at the site after the interaction. In some assays, an alternative to labeling the analytes and/or the analyte-bearing sites is so-called competitive assay. In a competitive assay, before, during or after the sample has been contacted with the reagent, analogs of the analytes are added to the sample, or to the reagent, or to the mixture of the sample and the reagent; and before, during or after such addition, the analogs and/or the analyte-bearing sites, are labeled with signal labels. In this specification, when reference is made to a signal label “associated” with an analyte or with an analyte-bearing site, the signal label can be one which identifies, in any way, interaction between an analyte and an analyte-interaction site, for example a signal label which is                (a) attached to an analyte or to an analyte-bearing site, or        (b) attached to an analyte analog, or to an analyte-bearing site, as part of a competitive assay.        
A representative sample of the particles is then examined, one particle at a time, in a cytometer (or like instrument) which recognizes the coding characteristic and the signal labels. On each particle, the coding characteristic identifies the analyte-interaction site, and the signal label(s) identifies (and usually quantifies) the analyte(s).
The coding characteristic on the particles is often provided by securing known amounts of one or both of two different fluorochromes to each particle. The cytometer (or other instrument) identifies the categories of particle by assessing the relative and/or absolute amounts of the fluorescence derived from the fluorochromes when they are exposed to a laser. Different categories of particles can alternatively or additionally be distinguished by other coding characteristics, for example by size, density, radioactivity, color, electrical charge, or magnetic properties.
The particles can be of different types, which are referred to herein as single-assay particles and multi-assay particles.
Single-assay particles contain analyte-interaction sites which can interact with (a) only one analyte, or (b) two or more analytes which do not need to be separately assayed on that particle. Associated assay particles are a particular class of single-assay particles. They contain analyte-interaction sites which can interact with all the analytes in a group of two or more analytes, and belong to two or more different categories, the number of categories being at least equal to the number of analytes in the group. The associated assay particles in each category contain analyte-interaction sites which are different from the analyte-interaction sites in each of the other categories. However, all the different analyte-interaction sites can interact with each analyte in the group of analytes, and the affinity of each of the analytes in the group for each of the analyte-interaction sites is known. The results of examining the associated assay particles in the different categories can, therefore, be analyzed together to assay each analyte.
Multi-assay particles contain analyte-interaction sites which interact with two or more analytes which must be separately assayed on each particle. Such particles require different signal labels to be associated with each of the different analytes. Multi-assay particles are usually dual-assay particles, i.e. they interact with only two different analytes. One important use of dual-assay particles is to assay two different types of rubella antibody, which may for example be labeled by different signal dyes before, during or after they interact with the particles.
Cytometers are often set up so that they can carry out both assays in which only single-assay particles are used and assays in which both single-assay and dual-assay particles are used.
It is generally convenient for dual-assay particles to use a pair of signal dyes which will fluoresce when exposed to the same laser, and for single-assay particles being assayed at the same time to use one of that pair of signal dyes, or another signal dye which will fluoresce when exposed to the same laser. However, such signal dyes generally fluoresce in spectra which have different peaks but which overlap substantially. This makes it difficult to assess separately the fluorescence produced by the respective signal dyes, especially when one of the signal dyes is present in much higher concentration than the other. The conventional practice is to split the fluorescence from the particle (whether it is a single-assay or dual-assay particle) into two relatively narrow and widely separated spectral bands. The fluorescence in one of the bands is derived principally from one of the signal dyes and the fluorescence in the other band is derived principally from the other signal dye. The accompanying FIG. 1 shows typical fluorescence spectra for a pair of signal dyes and the wavelength bands in which their fluorescence is conventionally assessed. As a result, only a fraction of the spectrum of each signal dye is assessed, and the sensitivity of the system is low. If the spectral bands are widened, the sensitivity of the system is increased, but the signal levels must be deconvolved mathematically to assign reliable values to each signal dye. This deconvolution requires knowledge of the spectra of the two signal dyes, and since these spectra may change in an unknown way in response to the microenvironment as the particle is being examined, errors may be introduced.
For disclosure of bead-based cytometry and similar assay procedures, reference may be made for example to U.S. Pat. Nos. 4,499,052 (Fulwyler), 4,665,020 (Saunders), 4,699,828 (Schwartz et al), 5,028,545 (Soini), 5,073,497 (Schwartz), 5,747,349 (van den Engh et al), 5,981,180 (Chandler et al.), 6,023,540 (Walt et al), 6,159,748 (Hechinger) and 6,165,796 (Bell), European Patent No. 126,450, WO 01/13120, and copending, commonly assigned, U.S. application Ser. No. 09/991,001, filed Nov. 14, 2001, the entire disclosures of which are incorporated by reference herein for all purposes.